Summary: We have previously shown that LLC-MK2 cells expressing the first nonstructural protein in the dengue 2 genome, NS1, support enhanced growth of wildtype dengue 2 virus. Initial studies on NS1 expressing cells infected with dengue 2 showed that the NS1 expressing cells were resistant to virus induced apoptosis. Further studies on virus induced apoptosis in control and NS1 expressing cells show that NS1 expressing cells are resistant to apoptosis induced by C2-ceramide. Published reports show that C2-ceramide is involved with the apoptosis inhibiting protein Bcl 2. There is no affect on Bcl 2 RNA and protein expression in NS1 expressing cells after treatment with C2-ceramide or after dengue 2 virus infection. These results suggest that a different control point in the initiation of apoptosis is involved in the NS1 mediated block in apoptosis. We have continued our study on the role of the dengue virus NS4B protein in virus replication. We have previously shown that lethal mutations in NS4B cannot be complemented in trans using a cell line expressing dengue 2 NS4B. This suggests that NS4B must function in cis in the viral genome, and/or must form complexes with other dengue nonstructural proteins which cannot be made by NS4B provided in trans. Coimmunoprecipitation studies between NS4B and other viral nonstructural proteins show no direct interactions. We are currently developing clones to analyze coexpression of NS4B and other nonstructural proteins and the ability of these clones to complement a mutant with NS4B completely deleted. We have initiated a new collaborative study on the role of the dengue virus nonstructural protein, NS5, in RNA replication. NS5 has previoiusly been shown to be the RNA dependent RNA polymerase (RdRp) for flaviviruses. However, little is know about the specificity of or requirements for activity of this protein. We purified native NS5 protein and NS5 protein which contained a 6X his tag at either the amino- or carboxy-terminus of the protein open reading frame expressed in Sf9 cells from baculovirus recombinants. Purified protein was tested for RdRp activity using dengue specific and control RNA templates. Terminal transferase activity was seen for all templates, however template dependent elongation was seen only on dengue templates. Further biochemical analysis of dengue NS5 RdRp activity is underway.